BackgroundPorcine circovirus 2 causes different clinical syndromes resulting in a significant economic loss in the pork industry. Three pigs with unexplained cardiac and multi-organ inflammation that tested negative for PCV2 and other known porcine pathogens were further analyzed.MethodsHistology was used to identify microscopic lesions in multiple tissues. Metagenomics was used to detect viral sequences in tissue homogenates. In situ hybridization was used to detect viral RNA expression in cardiac tissue.ResultsIn all three cases we characterized the genome of a new circovirus we called PCV3 with a replicase and capsid proteins showing 55 and 35 % identities to the genetically-closest proteins from a bat-feces associated circovirus and were even more distant to those of porcine circovirus 1 and 2. Common microscopic lesions included non-suppurative myocarditis and/or cardiac arteriolitis. Viral mRNA was detected intralesionally in cardiac cells. Deep sequencing in tissues also revealed the presence of porcine astrovirus 4 in all three animals as well as rotavirus A, porcine cytomegalovirus and porcine hemagglutinating encephalomyelitis virus in individual cases.ConclusionThe pathogenicity and molecular epidemiology of this new circovirus, alone or in the context of co-infections, warrants further investigations.

Bovine respiratory disease (BRD) is a common health problem for both dairy and beef cattle, resulting in significant economic loses. In order to identify viruses associated with BRD, we used a metagenomics approach to enrich and sequence viral nucleic acids in the nasal swabs of 50 young dairy cattle with symptoms of BRD. Following deep sequencing, de novo assembly, and translated protein sequence similarity searches, numerous known and previously uncharacterized viruses were identified. Bovine adenovirus 3, bovine adeno-associated virus, bovine influenza D virus, bovine parvovirus 2, bovine herpesvirus 6, bovine rhinitis A virus, and multiple genotypes of bovine rhinitis B virus were identified. The genomes of a previously uncharacterized astrovirus and picobirnaviruses were also partially or fully sequenced. Using real-time PCR, the rates of detection of the eight viruses that generated the most reads were compared for the nasal secretions of 50 animals with BRD versus 50 location-matched healthy control animals. Viruses were detected in 68% of BRD-affected animals versus 16% of healthy control animals. Thirtyeight percent of sick animals versus 8% of controls were infected with multiple respiratory viruses. Significantly associated with BRD were bovine adenovirus 3 (P < 0.0001), bovine rhinitis A virus (P ‫؍‬ 0.005), and the recently described bovine influenza D virus (P ‫؍‬ 0.006), which were detected either alone or in combination in 62% of animals with BRD. A metagenomics and realtime PCR detection approach in carefully matched cases and controls can provide a rapid means to identify viruses associated with a complex disease, paving the way for further confirmatory tests and ultimately to effective intervention strategies. IMPORTANCEBovine respiratory disease is the most economically important disease affecting the cattle industry, whose complex root causes include environmental, genetics, and infectious factors. Using an unbiased metagenomics approach, we characterized the viruses in respiratory secretions from BRD cases and identified known and previously uncharacterized viruses belonging to seven viral families. Using a case-control format with location-matched animals, we compared the rates of viral detection and identified 3 viruses associated with severe BRD signs. Combining a metagenomics and case-control format can provide candidate pathogens associated with complex infectious diseases and inform further studies aimed at reducing their impact. Bovine respiratory disease (BRD) is the most common and costly problem in the cattle industry, accounting for 70 to 80% of morbidity and 40 to 50% of mortality in U.S. feedlots (1, 2). The cattle industry is one of the largest agricultural sectors of the United States economy, with approximately three-quarters of a million farms raising cattle. The annual costs of BRD have been estimated at over 1 billion dollars per year (3, 4).In BRD, bacterial infections are thought to be opportunistic infections precipitated by viral infections causing damage to t...

Next-generation sequencing (NGS) approaches rapidly produce millions to billions of short reads, which allow pathogen detection and discovery in human clinical, animal and environmental samples. A major limitation of sequence homology-based identification for highly divergent microorganisms is the short length of reads generated by most highly parallel sequencing technologies. Short reads require a high level of sequence similarities to annotated genes to confidently predict gene function or homology. Such recognition of highly divergent homologues can be improved by reference-free (de novo) assembly of short overlapping sequence reads into larger contigs. We describe an ensemble strategy that integrates the sequential use of various de Bruijn graph and overlap-layout-consensus assemblers with a novel partitioned sub-assembly approach. We also proposed new quality metrics that are suitable for evaluating metagenome de novo assembly. We demonstrate that this new ensemble strategy tested using in silico spike-in, clinical and environmental NGS datasets achieved significantly better contigs than current approaches.

Using viral metagenomics of brain tissue from a young adult crossbreed steer with acute onset of neurologic disease, we sequenced the complete genome of a novel astrovirus (BoAstV-NeuroS1) that was phylogenetically related to an ovine astrovirus. In a retrospective analysis of 32 cases of bovine encephalitides of unknown etiology, 3 other infected animals were detected by using PCR and in situ hybridization for viral RNA. Viral RNA was restricted to the nervous system and detected in the cytoplasm of affected neurons within the spinal cord, brainstem, and cerebellum. Microscopically, the lesions were of widespread neuronal necrosis, microgliosis, and perivascular cuffing preferentially distributed in gray matter and most severe in the cerebellum and brainstem, with increasing intensity caudally down the spinal cord. These results suggest that infection with BoAstV-NeuroS1 is a potential cause of neurologic disease in cattle.

We describe the metagenomics-derived feline enteric virome in the faeces of 25 cats from a single shelter in California. More than 90 % of the recognizable viral reads were related to mammalian viruses and the rest to bacterial viruses. Eight viral families were detected: Astroviridae, Coronaviridae, Parvoviridae, Circoviridae, Herpesviridae, Anelloviridae, Caliciviridae and Picobirnaviridae. Six previously known viruses were also identified: feline coronavirus type 1, felid herpes 1, feline calicivirus, feline norovirus, feline panleukopenia virus and picobirnavirus. Novel species of astroviruses and bocaviruses, and the first genome of a cyclovirus in a feline were characterized. The RNA-dependent RNA polymerase region from four highly divergent partial viral genomes in the order Picornavirales were sequenced. The detection of such a diverse collection of viruses shed within a single shelter suggested that such animals experience robust viral exposures. This study increases our understanding of the viral diversity in cats, facilitating future evaluation of their pathogenic and zoonotic potentials.

Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultaneous detection of any previously genetically described viral nucleic acids in clinical samples. Viral genome sequences can also inform on likely phenotypes including drug susceptibility or neutralization serotypes. In this study, different variables of the laboratory methods often used to generate viral metagenomics libraries on the efficiency of viral detection and virus genome coverage were compared. A biological reagent consisting of 25 different human RNA and DNA viral pathogens was used to estimate the effect of filtration and nuclease digestion, DNA/RNA extraction methods, pre-amplification and the use of different library preparation kits on the detection of viral nucleic acids. Filtration and nuclease treatment led to slight decreases in the percentage of viral sequence reads and number of viruses detected. For nucleic acid extractions silica spin columns improved viral sequence recovery relative to magnetic beads and Trizol extraction. Pre-amplification using random RT-PCR while generating more viral sequence reads resulted in detection of fewer viruses, more overlapping sequences, and lower genome coverage. The ScriptSeq library preparation method retrieved more viruses and a greater fraction of their genomes than the TruSeq and Nextera methods. Viral metagenomics sequencing was able to simultaneously detect up to 22 different viruses in the biological reagent analyzed including all those detected by qPCR. Further optimization will be required for the detection of viruses in biologically more complex samples such as tissues, blood, or feces.

Humans keep more than 80 million cats worldwide, ensuring frequent contacts with their viruses. Despite such interactions the enteric virome of cats remains poorly understood. We analyzed a fecal sample from a single healthy cat from Portugal using viral metagenomics and detected five eukaryotic viral genomes. These viruses included a novel picornavirus (proposed genus “Sakobuvirus”) and bocavirus (feline bocavirus 2), a variant of feline astrovirus 2 and sequence fragments of a highly divergent feline rotavirus and picobirnavirus. Feline sakobuvirus A represents the prototype species of a proposed new genus in the Picornaviridae family, distantly related to human salivirus and kobuvirus. Feline astroviruses (mamastrovirus 2) are the closest relatives of the classic human astroviruses (mamastrovirus 1), suggestive of past cross-species transmission. Presence of these viruses by PCR among Portuguese cats was detected in 13% (rotavirus), 7% (astrovirus), 6% (bocavirus), 4% (sakobuvirus), and 4% (picobirnavirus) of 55 feline fecal samples. Co-infections were frequent with 40% (4/10) of cats shedding more than one of these viruses. Our study provides an initial unbiased description of the feline fecal virome indicating a high level of asymptomatic infections. Availability of the genome sequences of these viruses will facilitate future tropism and disease association studies.

Viruses with small circular rep-encoding ssDNA (CRESS-DNA) genomes can infect a wide range of eukaryotic organisms ranging from mammals to fungi. The genomes of two novel CRESS-DNA viruses, a cyclovirus (CyCV-SL) and gemycircularvirus (GemyCVSL) were detected by deep sequencing of the cerebrospinal fluids of Sri Lankan patients with unexplained encephalitis. One and three out of 201 CSF samples (1.5%) from unexplained encephalitis patients tested by PCR were CyCV-SL and GemyCV-SL DNA positive respectively. Nucleotide similarity searches of pre-existing datasets revealed related genomes in feces from unexplained cases of diarrhea from Nicaragua and Brazil and in untreated sewage from Nepal. Whether the tropism of the cycloviruses and gemycircularviruses reported here include humans or their detection reflects production from other cellular sources in or on the human body remains to be determined.